Good ÄKTATM system practice

Cue Cards
1. Good ÄKTA system practice 2
2. Run-Essentials 3
3. Fraction collector F9 and F9-C operation 4
4. Comprehensive System Wash and cleaning 5
5. UNICORN system notifications 6
6. Troubleshooting 7
6.1 Conductivity signal, a useful troubleshooting tool 7
6.2 Pressure alarms 8
6.3 Backpressure contributions 8
6.4 Fluctuating pressure 9
6.5 Fluctuating UV and conductivity signal, examples 10
and solutions
6.6 Cleaning the pH electrode and UV flow cell 11
6.7 Air bubble in a flow cell 12
6.8 Broken connector tip stuck in valve 13
6.9 Tubing guide 13
7. Abbreviations and References 14

Good ÄKTA system practice Cue Cards 29109616 AA

1. Good ÄKTA system practice

 Keep the system and Fraction collector clean, both on the outside and the
inside.
 Use inlet filters and filtered buffers, preferably degassed buffers.
 For optimal results, use buffers, system and columns at ambient temperature
during a purification run.
 Centrifuge or filter the samples prior sample loading, unless columns that can
handle crude samples are used.
 Use the Comprehensive System Wash and cleaning regularly for optimal
performance of the whole instrument, including Fraction collector. See p. 5.
 To prevent cross-contamination and bacterial growth in the instrument,
perform a System cleaning in place (System CIP) after each run. See p. 5.
 Limit system exposure to harsh cleaning solutions to maximum 2 hours.
 Fill the system with 20% ethanol to minimize bacterial growth prior storage.
 Set up UNICORNTM system notifications to let the system remind you when
your action is needed. For example, let a message appear once a week stating:
“Time to change pump rinsing solution”. See p. 6.

For further details see ÄKTA avant User Manual and ÄKTA pure User Manual
(References on page 14).

Good ÄKTA system practice Cue Cards 29109616 AA 2

2. Run-Essentials
Before During After
1. Check the 20% ethanol pump rinsing solution. Change if it appears Watch out for: For short term storage:
opaque or if the solvent level in the container has decreased.
• Overpressure, see p. 8 1. Empty Fraction collector
2. Check all tubing to make sure no tubing is nicked.
• Fluctuating pressure, see p. 9 2. Perform a Comprehensive
3. Make sure all buffers have the same temperature as the system to System Wash and/or cleaning
minimize air bubble issues. • Fluctuating UV signal, see
p. 10 3. Clean used columns in order
4. Prime inlets, i.e. fill all used buffer inlets with liquid. to increase their lifetime and
• Leakage
prevent:
5. Purge pumps to remove air from the pump heads. tighten connectors
• sample contamination
6. Start a flow and tighten connectors if leakage is observed. • Last tube filled (upon • cross contamination
7. Calibrate the pH electrode (if used). fractionation) • protein precipitation
change tubes, see p. 4 • column clogging
8. Perform a System Preparation run to fill the entire flowpath with the
right buffer into the system (including the Fraction collector). Sources of sound during For long term storage:
operation:
9. Start a flow to check if signals from monitors look stable and realistic If the instrument is not going to
If not, see p. 7-12. • Air ventilation be used for a couple of days or
10. Wipe off the Fraction collector sensors (code reader & drop sync • UV monitor – when using longer, clean as above and then
2-3 wavelengths fill
sensor) with a tissue and fill the Fraction collector with the
• Pump movement • instrument
tubes/plates needed.
• columns
11. Attach needed column(s) and set correct column pressure –
• Mixer
• pH flow cell
if running manually. • Changing valve position
with storage solution
• Fraction collector
movements

For further details see ÄKTA avant User Manual and ÄKTA pure User Manual
(References on page 14).

Good ÄKTA system practice Cue Cards 29109616 AA 3

3. Fraction collector F9 and F9-C operation
How to... Steps Outcome
Set Fraction collector 1. Make sure there is no run ongoing and the instrument is in state READY. The Fraction collector content is updated and all cassettes are
2. Open and close the Fraction collector door. ready to be used. To view the content of the Fraction collector,
content
3. The Fraction collector performs a Full scan. in the System control module select
NOTE: The Fraction collector content does not reset after the method has View:Fraction Collector Content.
ended unless the door has been opened and closed.

Take out fractions during 1. Pause the run. Fractionation continues in the next tube/well.
2. Open the Fraction collector door.
a run or in the middle of
3. Remove the desired tubes.
scouting or method 4. Close the the Fraction collector door.
queue runs 5. The Fraction collector performs a Quick scan.
6. Press Continue.
NOTE: If deep well plates have been removed these need to be replaced
with deep well plates of the same type before continuing the run.

Take out cassettes during 1. Pause the run. Fractionation continues into the next tube/well.
2. Open the Fraction collector door.
a run or in the middle of
3. Remove the desired cassette(s).
scouting or method 4. Replace any removed cassette(s) with the same type (filled or empty).
queue runs 5. Close the Fraction collector door.
6. The Fraction collector performs a Quick scan.
7. Press Continue.

Change tubes/plates 1. Open the Fraction collector door. Fractionation continues in the first tube/well of the replaced
2. Remove all cassette(s) that has been fully utilized. cassette. Fractionation always starts in the lowest cassette
after Last tube filled
3. Replace any removed cassette(s) with the same type. position number (1-6).
message 4. Close the Fraction collector door.
5. The Fraction collector performs a Quick scan.
6. Press Continue.

NOTE: The fraction collector is specially designed to automatically detect the cassettes present in the fraction collector but also to maximize the usage of the tubes/ wells . i.e.
every tube in the fraction collector should be available for fractionation. The fraction collector keeps track of which tubes/wells have been used during the runs and it is
therefore not possible to manually start fractionation in a specific cassette position or in a specific row.

NOTE: There is a possibility to try out other deep-well plates then the ones approved by GE Healthcare, provided that the deep-well plate specifications are fulfilled. In some cases,
the automatic cassette scanning may need to be disabled in System Control/System Settings/Fraction collector/Cassette configuration to make them work. Make sure
to manually update the Fraction collector content in System settings every time the content is changed. For further details see ÄKTA avant User Manual and ÄKTA pure
User Manual (References on page 14).
Good ÄKTA system practice Cue Cards 29109616 AA 4
4. Comprehensive System Wash and cleaning
… contains both manual (blue) and automated (white) procedures. It is used to wash/clean the system flowpath, i.e. not any column.

Create UNICORN method(s)

In all relevant method phases select modules and parts to cover the entire
flowpath, for example:
• All A&B inlets
• All sample inlets
• All column positions to be washed
NOTE: Make sure no columns are attached.
• All outlets (including outlet to Fraction collector)
• Injection valve
• Fraction collector* (if present)

Method outline for Method outline for

System Wash System Cleaning
(Use the predefined method for (Use the predefined method for
System Preparation) System CIP)

Method settings Method settings

System Preparation – Water System CIP – Water

System Preparation – Buffer System CIP – 0.1-0.5 M NaOH

System CIP – Buffer**

System CIP – 20% ethanol

For further details see ÄKTA avant User Manual and ÄKTA pure User Manual * Wash of Fraction collector accumulator is included.
(References on page 14). ** If water is used instead of buffer, this step will take longer time.

Good ÄKTA system practice Cue Cards 29109616 AA 5

5. UNICORN system notifications
Automated maintenance notifications for the system can remind you
when your action is needed. Follow the steps below in order to set a new
notification. In this exemple an automatic notification for changing the
pump rinsing solution is set.
1. In the System Control module, select System:Maintenance Manager
to open the Maintenance Manager dialog.
2. In the Maintenance Manager dialog, click the New System
Notification.
Result: The NewNotification field appears.
3. In the NewNotification field:
a. Enter a name for the notification, e.g. “Change pump rinsing
solution.”
b. Select a time interval after which the notification will be issued,
e.g. one week.
c. Write a message in the message input field that will be shown
for the maintenance notification, e.g. “Please change the pump
rinsing solution with a freshly made 20% ethanol solution.”
d. Click Apply to save the changes and apply the notification
settings.
4. Handle the maintenance notification when the set time interval has
been reached:
• Click Acknowledge to reset the counter for a new maintenance
notification period.
• Click Ignore to close the dialog without action.
NOTE: The Maintenance Notification will be displayed each
time the System Control module is opened until the
notification is acknowledged.

Good ÄKTA system practice Cue Cards 29109616 AA 6

6. Troubleshooting
6.1 Conductivity signal – a useful troubleshooting tool

Check the gradient

performance

Check that the conductivity

signal is stable before
sample injection

Possible to identify when the

buffer has been completely
exchanged/washed out.

Note: The conductivity signal becomes distorted at higher salt concentration since the ion activity decreases when the concentration
increases, i.e. a different curvature than the theoretical one is therefore normal at high salt concentrations.

For further details see ÄKTA avant User Manual and ÄKTA pure User Manual
(References on page 14).
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6.2 Pressure alarms 6.3 Backpressure
contributions
How to minimize
Source Note
the contribution
A larger i.d. will decrease the back
Keep the tubing as short as
Tubing pressure but will have a negative
possible and optimize the i.d.
effect on resolution. See p. 13.

The inline filter will prevent particles

in the solutions from entering the
Inline filter Change the filter regularly. flow path and column. With time, the
filter will start to clog and the
pressure will increase.

Decrease the flow rate when Mixing different liquids, e.g., in a

Buffer/solution running high-viscosity gradient, can increase the viscosity
buffers/ solutions. and result in higher back pressure.

Decrease the flow rate when Viscosity increases at lower

Temperature
running at low temperature. temperature.

Dilute viscous samples or

To avoid over-pressure, ÄKTA pure
decrease the flow rate during
and ÄKA avant systems have
sample application. Remove
optional pressure-controlled sample
Sample the sample inlet filter and if
application, where the flow rate is
using the system pump to
decreased as the pressure increases
apply sample, bypass the
and vice versa.
mixer.

Clean the column. See column instructions for cleaning.

Column Do not use smaller beads or Smaller beads will give higher
column diameter than the resolution but also higher back
application requires. pressure.

For further details see ÄKTA avant User Manual and ÄKTA pure User Manual
(References on page 14).

Good ÄKTA system practice Cue Cards 29109616 AA 8

6.4 Fluctuating pressure

Air in pump, example

Diagnose malfunctioning check valves

Inlet check valve diagnostic test, ”air bubble test”:
• Run the pump at 0.5 ml/min.
• Lift the inlet tubing a few seconds to let a small air bubble in.
• Normal function: air bubble moves forward and then stops,
and so on, no backward movement.
• Malfunction in inlet check valve: air bubble moves backwards
* For 150 ml/min systems, buffers can also be placed next to the system if preferred. before stopping.
After test: Purge the pump and make sure that the air bubble is
not stuck in the pump.

For further details see chapter on Troubleshooting in ÄKTA avant User Manual
and ÄKTA pure User Manual (References on page 14).

Good ÄKTA system practice Cue Cards 29109616 AA 9

6.5 Fluctuating UV and conductivity signal,
examples and solutions
Air spikes in UV signal

min

Small air bubbles Small air bubbles

passing UV and No air bubbles passing UV and
cond flow cell cond flow cell

Large air segments passing the flow cells

Large air segments Large air segments Air bubble stuck Air bubble flushed
passing UV and No air bubbles passing UV and out with increased
in cond flow cell
cond flow cell cond flow cell flow rate

Good ÄKTA system practice Cue Cards 29109616 AA 10

6.6 Cleaning the pH electrode and UV flow cell

Component Contaminant Cleaning solution

pH electrode Salt deposits 0.1 M NaOH and 0.1 M HCl
Lipid deposits Detergent or organic solvent
Protein deposits 1% pepsin in 0.1 M HCl
UV flow cell Salt/lipid/protein deposits Detergent, such as Deconex™

For further details see ÄKTA avant User Manual and ÄKTA pure User Manual
(References on page 14).

Good ÄKTA system practice Cue Cards 29109616 AA 11

6.7 Air bubble in a flow cell

Is the
flow restrictor
offline?
Set the flow restrictor
inline and continue
with the run.

Bypass the column and increase

the flow rate to 5 ml/min.

Does
the detector signal
go back to original
level?
Continue
with the run.

Connect an extra small i.d. tubing

(see p. 13) to the used outlet to
Large air segments
passing UV and Air bubble stuck in Air compressed into liquid and flushed out increase the backpressure.
conductivity flow cell cond flow cell due to increased back pressure

Does
the detector signal
go back to original
level?
Remove the extra
tubing and continue
with the run.

Remove the extra tubing and

flush the flow cells with 100%
MeOH. (Make sure that the MeOH
For further details see ÄKTA avant User Manual and ÄKTA pure User Manual is not coming in contact with
(References on page 14). high salt concentration buffers.)

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6.8 Broken connector tip stuck in valve
Step Action
1 Find a torx T7 screwdriver.
2 Firmly press the tip of the screwdriver into the broken tip.
3 Unscrew the broken connector tip.

6.9 Tubing guide

• There are many different sizes and types of tubing that can be connected to a chromatography system.
• Tubing with a smaller i.d. holds less delay volume and will therefore generate less dilution of the protein peak. However, narrow
tubing increases the system pressure, especially when running at high flow rates.
• The tubing and system used should match the application needs.

Color of 10 cm tubing 100 cm tubing

i.d. Standard tubing with/when
tubing corresponds to generates1
0.13 mm Red 1.3 μl 24 MPa Generating high back pressure, which is useful e.g. during
0.25 mm Blue 4.9 μl 1.7 MPa troubleshooting and when “running in” a new pump piston seal
0.50 mm Orange 20 μl 0.11 MPa Running at low flow rate using ÄKTA avant 25 and ÄKTA pure 25
0.75 mm Green 44 μl 0.02 MPa ÄKTA avant 25, ÄKTA pure 25 and ÄKTA pure 150
1.0 mm Beige 78 μl 0.007 MPa ÄKTA avant 150 and ÄKTA pure 150
1.0 mm Transparent 78 μl 0.007 MPa Outlet tubing for ÄKTA avant and ÄKTA pure
1.6 mm Transparent 200 μl –2 Inlet tubing for ÄKTA avant 25 and ÄKTA pure 25
2.9 mm Transparent 660 μl –2 Inlet tubing for ÄKTA avant 150 and ÄKTA pure 150
1 For water at 10 ml/min and room temperature
2 Negligible pressure

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7. Abbreviations and
References
Abbreviations
CIP Cleaning In Place
i.d. inner diameter
RT Room Temperature

GE and GE monogram are trademarks of General Electric Company.

References ÄKTA and UNICORN are trademarks of General Electric Company or one of its subsidiaries.
Deconex is a trademark of Borer Chemie AG.
ÄKTA Laboratory-scale Chromatography Systems All other third party trademarks are the property of their respective owner.
Instrument Management Handbook, 29-0108-31
Any use of UNICORN is subject to GE Healthcare Standard Software End-User License Agreement
ÄKTA pure User Manual, 29-0206-55 for Life Sciences Software Products. A copy of this Standard Software End-User License Agreement
ÄKTA avant User Manual, 29-0351-84 is available on request.
UNICORN 6 © 2009-2014 General Electric Company.
© 2014 General Electric Company – All rights reserved.
First published June 2014
All goods and services are sold subject to the terms and conditions of sale of
the company within GE Healthcare which supplies them. A copy of these terms and conditions is
available on request. Contact your local GE Healthcare representative for the most current
For local office contact information, visit information.
www.gelifesciences.com/contact GE Healthcare Europe GmbH
Munzinger Strasse 5, D-79111 Freiburg, Germany
GE Healthcare Bio-Sciences AB GE Healthcare UK Limited
Björkgatan 30 Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK
GE Healthcare Bio-Sciences Corp.
751 84 Uppsala
800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327, USA
Sweden GE Healthcare Japan Corporation
Sanken Bldg. 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073, Japan
www.gelifesciences.com/AKTA

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